A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II

Abstract

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four "site 1" and six "site 2" analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. Insulin-like growth factor II (IGF-II)2 is a single-chain polypeptide with homology to IGF-I and insulin. Its 67 amino acids are arranged, like those counterparts in IGF-I, in four domains in the order B, C, A, and D from the N terminus ( The mitogenic and metabolic activities of the IGFs and insulin result from their interaction with the type 1 IGF receptor (IGF-1R) and/or the exon 11-(IR-A) and exon 11ϩ (IR-B) isoforms of the insulin receptor (IR). These class II receptor tyrosine kinases exist at the membrane as preformed disulfidelinked homodimers composed of two ␣ and two ␤ subunits in a ␤-␣-␣-␤ arrangement (reviewed in Refs. 9 -11). The IR and IGF-1R share between 41 and 84% sequence similarity that is most pronounced in their tyrosine kinase domains. Despite the homology in sequence and structure between these receptors, each exhibits distinct ligand binding preferences. The IGF-1R and IR bind their cognate ligands with high affinity (IGF-I and insulin, respectively). Both the IGF-1R and IR-A bind IGF-II with high affinity and are capable of mediating IGF-II action (12). Interestingly, the IR-B has a low affinity for IGF-II. The discerning factors for this isoform discrimination are largely undefined but may involve steric hindrance between the 12 amino acids encoded by exon 11 of IR-B and the IGF-II C domain.There are currently no structures of any of these ligand⅐receptor complexes. The binding of insulin to the IR is certainly the best characterized of these interactions. Insulin has two IR binding surfaces: the "site 1" binding surface lies within the insulin dimerization surface, whereas "site 2" overlaps its hexamer-forming surface. Insulin is proposed to crosslink opposite halves of the insulin receptor dimer through these two receptor binding surfaces (13,14). The stoichiometry of binding at physiological insulin concentrations is 1:1. However, each IR has two potential ligand binding pockets both also consisting of two ligand binding surfaces (site 1 and site 2 of one monomer and site 1Ј and site 2Ј of the other, which combine to give two identical binding pockets, site 1/2Ј and site 1Ј/2). This putative arrangement is consistent with the three-dimensional crystal structure of the IR ectodomain (15). Evidence from ligand binding studies suggests that insulin may cross-link only one pair of binding surfaces at a time, with binding of a second molecule to an unoccupied site acce...

All-Atom Structural Models for Complexes of Insulin-Like Growth Factors IGF1 and IGF2 with Their Cognate Receptor

“…However, two major mutagenesis efforts have been made to map out the site 2 residues of IGFs: (1) Gauguin et al assigned E9, D12, F16, D53, L54, and E58 as site 2 residues of IGF1, 28 and (2) Alvino et al suggested E12, D15, F19, L53, and L57 as site 2 residues of IGF2. 29 All of these residues are part of the site 2 contacts with the (F1-F2)′ loops in our structural models (Table 1), except for D53 (IGF1), which is also consistent with mutagenesis work, as Ala mutation of D53 does not change the binding affinity of IGF1 for IGF1R. Because we see partial or no contact of D15 (IGF2) with either F2′ or L1, our data leave open the possibility that D15 could be a site 1 residue, as also suggested by Alvino et al 29 This is conceivable based on our IGF1/IGF1RΔβ model because the homologous residue D12 of IGF1 stably interacts with a site 1 residue R59 on the L1 domain in this model.…”

“…However, there is strong indirect evidence from our models in favor of these IGF1R residues as site 2 participants because these (F1-F2)′ residues contact exactly those residues that have been already demonstrated by careful mutagenesis as site 2 contacts on each ligand (vide supra). 28,29 Role of the C-domain and the D-domain of IGF1/IGF2…”

“…Also unclear is the role of the IGF1 D-domain, as contrasting details related to the significance of this domain in ligand binding have been reported: deletion of the IGF1 D-domain negligibly affects IGF1R binding affinity, 26 yet Ala mutation of K65 and K68 compromises the binding affinity by 10-fold. 27 Exactly which residues of L2 and the type III fibronectin domains (F1-F2) interact with assigned site 2 residues of IGF1 28 and IGF2, 29 respectively, remains unestablished. The exact registry of the L1 domain residues of IGF1R with the site 1 residues of IGF1/IGF2 also remains elusive due in part to the fact that there have been so far no detailed structural models of IGF-bound IGF1R to test.…”

Insulin-like Growth Factor-II (IGF-II) and IGF-II Analogs with Enhanced Insulin Receptor-a Binding Affinity Promote Neural Stem Cell Expansion

“…IGF-II consists of four domains, B, C, A, and D (the B and A domains are similar to the corresponding domains in insulin) (13). These domains contain residues that are important in binding IGF-1R and IR.…”

“…Binding of ligand results in cross-linking of the two receptor halves leading to a structural change and subsequent activation of the tyrosine kinase domain. The residues on IGF-II important for binding the IR site 1 include Val-43, Phe-26, and Tyr-27, whereas residues Glu-12, Phe-19, Leu-53, and Glu-57 contact site 2 (13). In this study, we used modified forms of IGF-II that contained mutations at these sites of interaction.…”